C. neoformans is a significant fungal pathogen, particularly in immunocompromised patients. It is important to understand the biosynthesis and regulation of fungal cell wall, especially because they are essential organelles that have been successfully used as targets for antifungal therapy. In our previous funding period we have generated significant preliminary data to suggest that the protein kinase C (PKC1) pathway impacts cell wall integrity and virulence in C. neoformans and have identified mutants that have weakened cell walls and that are avirulent or are hypervirulent. Additionally, we have shown that PKC1 has a role in response to oxidative and nitrosative stresses. In this application, we propose to further characterize the PKC1 pathway to determine which genes are important for signaling and cell wall integrity, to define the proteins that interact with the PKC1 pathway in C. neoformans, to determine the downstream genes that are affected by perturbations in the pathway, and to identify the proteins that sense cell wall damage. There are three specific aims. In the first, we will determine the downstream effects of perturmations in the pathway using micro-arrays and biochemical analysis. We will identify genes that are miss-regulated in our mutants, with the goal of being able to establish the specific defects in the cell walls of the mutants. In the second aim, we will dissect the role of the upstream regulators of the pathway, specifically the Rho proteins and the guanine exchange factors and the GTPase activating proteins. Our preliminary data indicates that this part of the pathway is distinct from S. cerevisiae. In the third aim, we will employ a genetic screen to find the proteins that act as sensors for the PKC1 pathway with an emphasis on oxidative and nitrosative stress, and trigger activation of the pathway. In this application we have proposed to analyze the PKC1 pathway in the fungal pathogen, C. neoformans. This pathway is critical for cell growth, resistance to cell wall damaging agents and for virulence, and is induced in response to antifungal agents. For these reasons, it is critical to delineate the components of the pathway, determine how they interact, what cellular effects the pathway has, and how the pathway receives input from the environment. These studies should elucidate the mechanisms that fungal cells use to regulate biosynthesis of their walls, with the long-term goal of identifying novel targets for antifungal therapy. PUBLIC HEALTH RELEVANCE: Fungal infections have become more prevalent in recent years due to the increase in the immunocompromised patient population from AIDS, organ transplants and chemotherapies. Systemic fungal infections are serious health threats, and safe, highly effective antifungal therapies are not available. Biosynthesis of the fungal cell wall is an attractive target for antifungal therapies because the cell wall is an essential organelle that is not present in the human host, and this project will delineate signal transduction that determines the biosynthesis of the cell wall of a fungal pathogen, Cryptococcus neoformans.